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Epidemiological studies indicate that obesity, insulin resistance, and diabetes are important comorbidities of patients with ischemic heart disease and increase mortality and development of congestive heart failure after myocardial infarction. Although ob/ob and db/db mice are commonly used to study obesity with insulin resistance or diabetes, mutations in the leptin gene or its receptor are rarely the cause of obesity in humans, which is, instead, primarily a consequence of dietary and lifestyle factors. Therefore, we used a murine model of diet-induced obesity to examine the physiological effects of obesity and the inflammatory and healing response of diet-induced obese (DIO) mice after myocardial ischemia-reperfusion injury. DIO mice developed hyperinsulinemia and insulin resistance and hepatic steatosis, with significant ectopic lipid deposition in the heart and cardiac hypertrophy in the absence of significant changes in blood pressure. The mRNA levels of chemokines at 24 h and cytokines at 24 and 72 h of reperfusion were higher in DIO than in lean mice. In granulation tissue at 72 h of reperfusion, macrophage density was significantly increased, whereas neutrophil density was reduced, in DIO mice compared with lean mice. At 7 days of reperfusion, collagen deposition in the scar was significantly reduced and left ventricular (LV) dilation and cardiac hypertrophy were increased, indicative of adverse LV remodeling, in infarcted DIO mice. Characterization of a murine diet-induced model of obesity and insulin resistance that satisfies many aspects commonly observed in human obesity allows detailed examination of the adverse cardiovascular effects of diet-induced obesity at the molecular level. insulin resistance; cardiac lipotoxicity; ischemia-reperfusion injury; left ventricular remodeling

We have developed a high-frequency, high-resolution Doppler spectrum analyzer (DSPW) and compared its performance against an adapted clinical Medasonics spectrum analyzer (MSA) and a zero-crossing interval histogram (ZCIH) used previously by us to evaluate cardiovascular physiology in mice. The aortic velocity (means [+ or -] SE: 92.7 [+ or -] 2.5 versus 82.2 [+ or -] 1.8 cm/s) and aortic acceleration (8194 [+ or -] 319 versus 5178 [+ or -] 191 cm/[s.sup.2]) determined by the DSPW were significantly higher compared to those by the MSA. Aortic ejection time was shorter (48.3 [+ or -] 0.9 versus 64.6 [+ or -] 1.8 ms) and the isovolumic relaxation was longer (17.6 [+ or -] 0.6 versus 13.5 [+ or -] 0.6 ms) when determined by the DSPW because it generates shorter temporal widths in the velocity spectra when compared to the MSA. These data indicate that the performance of the DSPW in evaluating cardiovascular physiology was better than that of the MSA. There were no significant differences between the aortic pulse wave velocity determined by using the ZCIH (391 [+ or -] 16 cm/s) and the DSPW (394 [+ or -] 20 cm/s). Besides monitoring cardiac function, we have used the DSPW for studying peripheral vascular physiology in normal, transgenic, and surgical models of mice. Several applications such as the detection of high stenotic jet velocities (>4 m/s), vortex shedding frequencies (250 Hz), and subtle changes in wave shapes in peripheral vessels which could not obtained with clinical Doppler systems are now made possible with the DSPW. Index Terms--High-frequency pulsed Doppler ultrasound, mouse cardiac function, mouse cardiovascular physiology, pulse-wave velocity, stenotic jet velocities, vortex shedding frequencies.

Limitation of damage after ischemia and reperfusion injury to the myocardium remains an elusive clinical goal. Previous studies have suggested that molecular chaperones, which include members of the heat shock protein (Hsp) family, may have cardioprotective effects, although the protective role of endogenous chaperones has not been well documented. CHIP (carboxyl terminus of Hsp70-interacting protein) is a cochaperone/ubiquitin ligase that integrates the response to stress at multiple levels. We tested the response of CHIP(-/-) mice to in vivo ischemia and reperfusion injury induced by left anterior descending coronary artery ligation. Compared with wild-type littermates, CHIP(-/-) mice had decreased survival and increased incidence of arrhythmias during reperfusion. The size of myocardial infarction, as assessed by the ratio of infarct area to area at risk, was 50% greater in CHIP(-/-) mice. Increased infarct size was accompanied by impaired upregulation of the chaperone Hsp70 after ischemia-reperfusion injury. In situ analysis also indicated that hearts of CHIP(-/-) mice were more prone to develop apoptosis in cardiomyocytes and especially endothelial cells of intramural vessels. Previous studies have found that CHIP plays a central role in maintaining protein quality control and coordinating the response to stress. The present data indicate that these functions of CHIP provide a critical cardioprotective effect in the setting of ischemia-reperfusion injury due in part to increased apoptosis in cardiac cells. Quality control mechanisms therefore may be underappreciated clinical targets for maximizing myocardial protection after injury. chaperone; heat shock protein; apoptosis

Despite the extensive use of genetically altered mice to study cardiovascular physiology and pathology, it remains difficult to quantify arterial function noninvasively in vivo. We have developed a noninvasive Doppler method for quantifying vessel wall motion in anesthetized mice. A 20-MHz probe was held by an alligator clip and positioned over the carotid arteries of 16 mice, including six 3- to 5-mo-old wild-type (WT), four 30-mo-old senescent (old), two apolipoprotein E null (ApoE), and four [alpha]-smooth muscle actin null ([alpha]-SMA) mice. Doppler signals were obtained simultaneously from both vessel walls and from blood flow. The calculated displacement signals from the near and far walls were subtracted to generate a diameter signal from which the excursion and an augmentation index were calculated. The excursion ranged between 13 [micro]m (in ApoE) and 95 [micro]m (in [alpha]-SMA). The augmentation index was lowest in the WT mice (0.06) and highest in the old mice (0.29). We conclude that Doppler signal processing may be used to measure vessel wall motion in mice with high spatial and temporal resolution and that diameter signals can replace pressure signals for calculating the augmentation index. This noninvasive method is able to identify and confirm characteristic changes in arterial properties previously associated with age, atherosclerosis, and the absence of vascular tone. Doppler displacement; arterial mechanics; blood velocity; carotid artery

Mice are used with increasing frequency as models of human cardiovascular diseases, but significant gaps exist in our knowledge of vascular function in the aging mouse. We determined aortic input impedance spectra, pulse wave velocity, and augmentation index in adult (8-mo-old) and old (29-mo-old) mice to determine whether arterial stiffening occurred with age in mice as it does in humans. Pressure and blood velocity signals measured simultaneously from the same location in the ascending aorta were used to determine input impedance spectra (0-10 harmonics). The first minimum of the impedance modulus occurred at the second harmonic in adult mice but shifted to the fourth harmonic in old mice. Characteristic impedance (average of 2nd-10th harmonic) was 57% higher in old mice: 471 [+ or -] 62 vs. 299 [+ or -] 10 (SE) dyn.s.[cm.sup.-3] (P < 0.05). Pulse pressure and augmentation index, determined from the aortic pressure signals, were also higher in old mice: 42 [+ or -] 2.2 vs. 29 [+ or -] 4.9 mmHg (P < 0.05) and 37 [+ or -] 5 vs. 14 [+ or -] 2% (P < 0.005). Aortic pulse wave velocity measured from the timing of upstrokes of the Doppler velocity signals was 45% higher in old mice: 416 [+ or -] 22 vs. 286 [+ or -] 14 cm/s (n = 3, P < 0.01). These results reproduce age-related findings reported in humans and confirm that mice may be used as models of age-related vascular stiffening. aging mouse; arterial stiffness; characteristic impedance; pulse wave velocity; augmentation index

Myocardial infarction is associated with the rapid induction of mononuclear cell chemoattractants that promote monocyte infiltration into the injured area. Monocyte-to-macrophage differentiation and macrophage proliferation allow a long survival of monocytic cells, critical for effective healing of the infarct. In a canine infarction-reperfusion model, newly recruited myeloid leukocytes were markedly augmented during early reperfusion (5-72 h). By 7 days, the number of newly recruited myeloid cells was reduced, and the majority of the inflammatory cells remaining in the infarct were mature macrophages. Macrophage colony-stimulating factor (MCSF) is known to facilitate monocyte survival, monocyte-to-macrophage conversion, and macrophage proliferation. We demonstrated marked induction of MCSF mRNA in ischemic segments persisting for at ]east 5 days after reperfusion. MCSF expression was predominantly localized to mature macrophages infiltrating the infarcted myocardium; the expression of the MCSF receptor, c-Fms, a protein with tyrosine kinase activity, was found in these macrophages but was also observed in a subset of microvessels within the infarct. Many infarct macrophages expressed proliferating cell nuclear antigen, a marker of proliferative activity. In vitro MCSF induced monocyte chemoattractant protein-1 synthesis in canine venous endothelial cells. MCSF-induced endothelial monocyte chemoattractant protein-1 upregulation was inhibited by herbimycin A, a tyrosine kinase inhibitor, and by LY-294002, a phosphatidyl-inositol 3'-kinase inhibitor. We suggest that upregulation of MCSF in the infarcted myocardium may have an active role in healing not only through its effects on cells of monocyte/ macrophage lineage, but also by regulating endothelial cell chemokine expression. inflammation; reperfusion; chemokine; growth factors; macrophage colony-stimulating factor

Entman. Heart failure and greater infarct expansion in middle-aged mice: a relevant model for postinfarction failure. Am J Physiol Heart Circ Physiol 282: H615-H621, 2002; 10.1152/ajpheart.00206.2001.--Young mice tolerate myocardial loss after coronary artery ligation (CAL) without congestive heart failure (CHF) signs or mortality. We predicted a CHF phenotype after CAL in aged mice. Left coronary artery ligation produced permanent myocardial infarcts (MI). Mortality was higher in male 14-mo-old C57BL/6N mice (Older mice) than in 2-mo-old mice (Young mice) (16 of 25 Older mice died vs. 0 of 10 Young mice, P < 0.02). After 8 wk, rales, weight loss, and lethargy preceded deaths. Captopril (50 mg * [kg.sup.-1] * [day.sup.-1]) increased Older mouse survival (6 of 22 died, P < 0.02). Captopril improved systolic function (peak aortic blood velocity) from 76 [+ or -] 6% of baseline in untreated Older mice to 93 [+ or -] 8% (P < 0.036). At 24 h, MI comprised 28 [+ or -] 4% of the left ventricle in Young mice, surprisingly larger than that in Older mice (18 [+ or -] 2%, P < 0.011). Endocardial area underlying the infarct scar was significantly larger in Older mice than in Young mice. Captopril did not reduce expansion but markedly reduced septal hypertrophy. Aging reduces compensatory ability in mice despite smaller acute infarcts. Less effective myocardial repair, greater infarct expansion, and septal hypertrophy are seen with aging. Aging is a more relevant murine model of post-MI heart failure in patients. heart failure; myocardial infarction; aging; mouse

Nossuli, Tareck O., Nikolaos G. Frangogiannis, Pascal Knuefermann, Venkatesh Lakshminarayanan, Oliver Dewald, Alida J. Evans, Jacques Peschon, Douglas L. Mann, Lloyd H. Michael, and Mark L. Entman. Brief murine myocardial I/R induces chemokines in a TNF[alpha]-independent manner: role of oxygen radicals. Am J Physiol Heart Circ Physiol 281: H2549-H2558, 2001.--Early chemokine induction in the area at risk of an ischemic-reperfused (I/R) myocardium is first seen in the venular endothelium. Reperfusion is associated with several induction mechanisms including increased extracellular tumor necrosis factor (TNF)-[alpha], reactive oxygen intermediate (ROI) species formation, and adhesion of leukocytes to the venular endothelium. To test the hypothesis that chemokine induction in cardiac venules can occur by ROIs in a TNF-[alpha]-independent manner, and in the absence of leukocyte accumulation, we utilized wild-type (WT) and TNF-[alpha] double-receptor knockout mice (DKO) in a closed-chest mouse model of myocardial ischemia (15 rain) and reperfusion (3 h), in which there is no infarction. We demonstrate that a single brief period of I/R induces significant upregulation of the chemokines macrophage inflammatory protein (MIP) -1[alpha], -1[beta], and -2 at both the mRNA and protein levels. This induction was independent of TNF-[alpha], whereas levels of these chemokines were increased in both WT and DKO mice. Chemokine induction was seen predominantly in the endothelium of small veins and was accompanied by nuclear translocation of nuclear factor-[kappa]B and c-Jun (AP-l) in venular endothelium. Intravenous infusion of the oxygen radical scavenger N-2-mercaptopropionyl glycine (MPG) initiated 15 min before ischemia and maintained throughout reperfusion obviated chemokine induction, but MPG administration after reperfusion had begun had no effect. The results suggest that ROI generation in the reperfused myocardium rapidly induces C-C and C-X-C chemokines in the venular endothelium in the absence of infarction or irreversible cellular injury. macrophage inflammatory protein; endothelium; transgenic animals Received 9 February 2001; accepted in final form 22 August 2001

Two hours of occlusion followed by reperfusion or permanent left anterior descending coronary artery occlusion in C57BL/6 mice were examined for anatomic and functional changes. About 30% of the left ventricle had infarcts generated by both interventions measured at 24 h and equivalent suppression of LV ejection velocity and filling velocity measured by Doppler ultrasound at 1 wk. The results showed that later reperfusion appeared to reduce the degree of infarct expansion.

Researchers have investigated whether noninvasive Doppler velocimetry using high-speed signal processing can provide signals from which aortic pulse-wave velocity (PWV) can be determined in mice. This involved using a custom-made 20-MHz pulsed Doppler instrument to obtain blood flow velocity signals from the aortic arch and the abdominal aorta. It was found that arterial tonometry using micromanometers can be used to obtain details of high-fidelity pressure waveforms from mice without arterial cannulation.

The noninvasive pulsed Doppler ultrasound measurements of cardiac systolic and diastolic function in mice give results similar to those of larger species with respect to magnitude and response to hyperthyroidism and aging. The induction of hyperthyroidism results in an increase in systolic and diastolic function in mouse. Diastolic function is accompanied by ATP-dependent Ca2+ ion uptake. The sequestration of calcium ions to sarcoplasmic reticulum controls the cardiac relaxation.

Genetically altered mice and unique experimental procedures involving coronary occlusion were developed for the study of the inflammatory reactions following myocardial ischemia-reperfusion. The procedures allow the assessment of cardiac function, area at risk and infarct size. These procedures combined with transgenic mice overexpressing or deficient in certain genes would allow the assessment of the role of specific gene products in the pathophysiology of inflammation and myocardial injury following ischemia-reperfusion.

Audio-Doppler signal recording from the ascending aorta of mice using a 20-MHz transducer helps assess the utility of 20-MHz pulsed Doppler ultrasound in monitoring cardiovascular parameters in small animals. Playback of the audio signals into a fast Fourier transform analyzer helps measure ejection time, heart rate, mean and peak velocities, stroke distance, rise time and acceleration from the spectral envelopes. Aortic velocity signals are obtained through moistened fur using the transducer, indicating that 20-MHz ultrasound can be successfully applied for the noninvasive measurement of aortic velocity in small animals.

Adult and neonatal cardiac sarcolemmal preparations from dogs were used in the determination of postnatal developmental changes in transporter function and density. Results reveal that the density of sodium-potassium pumpschanges with age. In contrast, the sodium-calcium exchanger does not exhibit age-related alterations. This implies that calcium absorption through the sodium-calcium exchanger is more significant in neonates than in adults.

Leukocyte trafficking and myocardial reperfusion injury in ICAM-1/P-selectin-knockout mice. Am J Physiol Heart Circ Physiol 280: H60-H67, 2001.--P-selectin and intercellular adhesion molecule-1 (ICAM-1) mediate early interaction and adhesion of neutrophils to coronary endothelial cells and myocytes after myocardial ischemia and reperfusion. In the present study, we examined the physiological consequences of genetic deletions of ICAM-1 and P-selectin in mice. In wild-type mice, after 1 h of ischemia followed by reperfusion, neutrophil influx into the area of ischemia was increased by 3 h with a peak at 24 h and a decline by 72 h. ICAM-1/P-selectin-deficient mice showed a significant reduction in neutrophils by immunohistochemistry or by myeloperoxidase activity at 24 h but no significant difference at 3 h. Infarct size (area of necrosis/area at risk) assessed 24 h after reperfusion was not different between wild-type and deficient mice after 30 min and 1 h of occlusion. Mice with a deficiency in both ICAM-1 and P-selectin have impaired neutrophil trafficking without a difference in infarct size due to myocardial ischemia-reperfusion. cell adhesion molecules; intercellular adhesion molecule 1; inflammation; neutrophils

Hartley, Craig J., Anilkumar K. Reddy, Sridhar Madala, Baby Martin-McNulty, Ronald Vergona, Mark E. Sullivan, Meredith Halks-Miller, George E. Taffet, Lloyd H. Michael, Mark L. Entman, and YiXin Wang. Hemodynamic changes in apolipoprotein E-knockout mice. Am J Physiol Heart Circ Physiol 279: H2326-H2334, 2000.--Apolipoprotein E-knockout (ApoE-KO) mice develop advanced atherosclerotic lesions by 1 yr of age and have been well characterized pathologically and morphologically, but little is known regarding their cardiovascular physiology and hemodynamics. We used noninvasive Doppler ultrasound to measure aortic and mitral blood velocity and aortic pulse-wave velocity in 13-mo-old ApoE-KO and wild-type (WT) mice anesthetized with isoflurane. In other mice from the same colony, we measured systolic blood pressure, body weight, heart weight, cholesterol, and hematocrit. Heart rate and blood pressure were comparable (P = not significant) between ApoE-KO and WT mice, but significant decreases (P [is less than] 0.001) were found in body weight (-22%) and hematocrit (-11%), and significant increases were found in heart weight (+23%), aortic velocity (+60%), mitral velocity (+81%) (all P [is less than] 0.001), and pulse-wave velocity (+ 13%, P [is less than] 0.05). We also found inflections in the aortic arch velocity signal consistent with enhanced peripheral wave reflection. Thus ApoE-KO mice have phenotypic alterations in indexes of peripheral vascular resistance and compliance ,and significantly elevated cardiac outflow velocities and heart weight-to-body weight ratios. atherosclerosis; cardiac output; hypertrophy; ultrasonics